UC-NRLF 


STUDIES  IN  SPERMATOGENESIS 


WITH  ESPECIAL  REFERENCE  TO  THE 


"ACCESSORY  CHROMOSOME" 


BY  N.  M.  STEVENS. 


WASHINGTON,  D.  C.: 

Published  by  the  Carnegie  Institution  of  Washington 
September,  1905. 


STUDIES  IN  SPERMATOGENESIS 


WITH   ESPECIAL  REFERENCE  TO  THE 


"ACCESSORY  CHROMOSOME" 


BY  N.  M.  STEVENS. 


WASHINGTON,  D.  C.: 

Published  by  the  Carnegie  Institution  of  Washington 
September,  1905. 


CARNEGIE  INSTITUTION  OF  WASHINGTON 
PUBLICATION  No.  36 


FROM  THE  PRESS  OF 

THE  HENRY  E.  WILKENS  PRINTING  CO. 

WASHINGTON,  D.  C. 


STUDIES  IN  SPERMATOGENESIS  WITH  ESPECIAL  REF- 
ERENCE TO  THE  "ACCESSORY  CHROMOSOME." 


By  N.  M.  STEVENS. 

In  connection  with  the  problem  of  sex  determination  it  has  seemed 
necessary  to  investigate  further  'the  so-called  "  accessory  chromo- 
some," which,  according  to  McClung  ('02),  may  be  a  sex  determinant. 
This  view  has  been  supported  by  Button  ('02)  in  his  work  on  Brachy- 
stola  magnet,  but  rejected  by  Miss  Wallace  ('05)  for  the  spider. 

The  forms  selected  for  study  have  been  taken  from  several  groups 
of  insects,  and  are  all  species  whose  spermatogenesis  has  not  been 
previously  worked  out.  They  are  (i)  a  California  termite,  Termopsis 
angusticollis ;  (2)  a  California  sand-cricket,  Stenopelmatus ;  (3)  the 
croton-bug,  Blatlella  germanica ;  (4)  the  common  meal-worm,  Tene- 
brio  molitor ;  and  (5)  one  of  the  aphids,  Aphis  cenothercz. 

A  brief  account  of  a  chromatin  element  resembling  the  accessory 
chromosome  in  Sagitta  has  been  added  for  comparison.  The  spermato- 
genesis of  each  form  will  be  described  in  detail,  and  a  general  discussion 
of  the  results  and  their  relation  to  the  accessory  chromosome  and  sex 
determination  will  follow.  The  spermatogenesis  of  the  aphid  has  been 
included  in  another  paper,  but  a  summary  of  results  and  a  few  figures 
will  be  given  here  for  reference  in  the  general  discussion. 

METHODS. 

The  testes  were  fixed  in  various  fluids — Flemming's  strong  solution, 
Hermann's  platino-aceto-osmic,  Gilson's  mercuro-nitric,  Lenhossek's 
alcoholic  sublimate  acetic,  and  corrosive  acetic.  Flemming's  and 
Hermann's  fluids  followed  by  safranin  gave  good  results  in  most 
cases.  The  mercuro-nitric  solution  and  Lenhossek's  fluid  gave  excel- 
lent fixation  and  were  preferable  to  the  osmic  mixtures  when  it  was 
desirable  to  stain  the  same  material  with  iron-hsematoxylin,  and  also 
with  various  anilin  stains. 

Heidenhain's  iron-haematoxylin,  either  alone  or  with  orange  G 
or  erythrosin,  was  used  more  than  any  other  one  stain.  With  osmic 
fixation  safranin  gave  better  results  in  some  cases,  because  of  the 

3 


4  STUDIES    IN    SPERMATOGENESIS. 

abundance  of  spindle  fibers  and  sphere  substance  which  were  stained  by 
haematoxylin .  The  safranin -gentian  combination  used  by  Miss  Wal- 
lace and  others  in  the  study  of  the  accessory  chromosome  did  not  prove 
to  be  especially  helpful  with  these  forms.  Thionin  was  found  to  be  a 
very  useful  stain  for  distinguishing  between  the  accessory  chromosome 
and  an  ordinary  nucleolus.  Lacht-griin  was  often  used  in  combination 
with  safranin. 

RESULTS  OF  INVESTIGATIONS. 

Termopsis  angusticollis. 

In  the  termite  it  was  not  found  to  be  practicable  to  dissect  out  the 
testes.  The  tip  of  the  abdomen  was  therefore  fixed  and  sectioned, 
young  males  whose  wings  were  just  apparent  being  used.  The  cells 
are  all  small,  and  could  not  be  studied  to  advantage  with  less  than 
1500  magnification  (Zeiss  oil  immersion  2  mm.,  oc.  12). 

In  the  spermatogonium  there  is  a  very  large  nucleolus  (plate  i, 
fig.  i),  which  in  the  iron-hsematoxylin  preparations  is  very  conspic- 
uous, but  does  not  stain  like  chromatin  with  thionin  or  other  anilin 
stains,  nor  does  it  behave  like  an  accessory  chromosome  during  the 
maturation  mitoses.  Before  each  spermatogonial  division  it  divides 
as  in  figures  2  and  3,  and  the  same  is  true  for  each  maturation  mitosis. 
Figure  4  shows  the  52  chromosomes  of  a  spermatogonial  division  in 
metaphase.  Figures  5  and  6  are  young  spermatocytes,  showing  the 
division  of  the  nucleolus.  Figures  8,  9,  and  10  show  a  stage  imme- 
diately following  that  shown  in  figure  6  and  evidently  persisting  for 
some  time.  The  spireme  thread  is  very  fine,  stains  deeply,  and  is 
wound  into  a  dense  ball,  often  concealing  one  (fig.  10)  or  both  nucleoli 
(fig.  8).  Figure  n  shows  the  next  stage  ;  the  bivalent  chromosomes 
are  so  disposed  as  to  give  the  familar  "  bouquet  stage,"  with  the  loops 
directed  away  from  the  centrosome  and  sphere  (c).  Figures  12,  13, 
and  14  show  the  later  development  of  the  same  stage,  the  chromatin 
loops  becoming  thicker  by  the  concentration  of  the  smaller  granules 
to  form  the  larger  ones  seen  in  figure  14.  The  loops  now  straighten 
out  and  extend  in  various  directions  across  the  nuclear  space  (figs.  15, 
16,  17).  In  fig.  18  a  a  longitudinal  split  is  seen  in  several  chromo- 
somes. Figures  i8£,  19,  20,  and  21  show  various  stages  in  the  con- 
traction of  these  split  bivalent  chromosomes  to  form  diamond-shaped 
tetrads,  each  side  of  which  is  a  univalent  daughter  chromosome.  The 
tetrads  come  into  the  spindle  in  this  form  (figs.  22,  23),  and  change  to 
the  form  shown  in  figure  24  during  the  metaphase  (figs.  22,  26,  28). 
Figures  25  and  27  show  the  26  bivalent  chromosomes,  or  tetrads,  in 


TERMOPSIS   ANGUSTICOLLIS.  5 

early  and  late  metaphase,  respectively,  and  figures  29,  30,  and  31  in 
anaphase.  This  is  certainly  a  reduction  division,  for  the  tetrads  are 
always  somewhat  elongated  and  come  into  the  spindle  with  their  longer 
axes  parallel  with  the  axis  of  the  spindle.  The  aberrant  bodies  in 
these  figures  are  probably  remains  of  the  nucleoli ;  they  are  found  only 
in  iron-hsematoxylin  preparations.  Figures  31  and  32  show  excep- 
tional cases  where  the  cell  has  divided.  Usually  the  two  daughter 
nuclei  are  formed  in  an  undivided  cell.  The  resting-stage  between 
the  two  divisions  is  only  partial .  The  nucleolus  appears  and  divides 
into  two  (figs.  33-36),  and  the  chromosomes  change  into  the  dyad 
form  (fig.  36),  in  which  they  come  into  the  second  maturation  spindle 
(figs.  37,  38).  The  equatorial  plate  again  shows  26  chromosomes  (fig. 
39) ,  The  formation  of  the  spermatozoa  is  peculiar  in  that  the  original 
spermatocyte  cell -body,  as  a  rule,  does  not  divide  ;  but  the  four  nuclei 
resulting  from  the  two  maturation  divisions  develop  into  sperm-heads 
in  one  cell.  All  have  a  nucleolus  (fig.  41),  and  in  a  slightly  later  stage 
(fig.  42)  the  elongated  nuclei  have  a  distinct  centrosome  and  sphere  at 
the  posterior  end.  L,ater  stages  are  shown  in  figures  43,  44,  and  45. 

The  points  of  greatest  interest  in  the  spermatogenesis  of  Termopsis 
angusticollis  are,  (i)  the  fact  that  no  accessory  chromosome  is  present ; 
(2)  that  the  method  of  tetrad  formation  and  reduction  are  clear,  despite 
the  fact  that  the  cells  and  the  chromatin  elements  are  quite  small ; 
and  (3)  the  failure  of  the  cell-bodies  to  divide  and  the  consequent 
development  of  four  spermatozoa  in  one  cell. 

Stenopelmatus. 

The  spermatogonium  of  Stenopelmatus  contains  from  one  to  three 
large  nucleoli,  which  stain  much  less  with  thionin  than  does  the 
spireme  (plate  11,  figs.  46,47,  48).  As  the  distinct  chromosomes  come 
into  view  in  the  prophase  of  mitosis,  two  are  seen  to  be  nearly  twice 
as  long  as  the  others,  but  of  equal  length  (figs.  48,  49,  50.)  There 
are  46  chromosomes  in  the  equatorial  plate  of  a  spermatogonial  spindle 
(fig  50).  Besides  the  nucleolus  (n),  there  appears  in  the  young  sperm- 
atocyte a  conspicuous  element  (x)  which  stains  deeply  with  all  chro- 
matin stains  (fig.  51).  It  is  closely  applied  to  the  nuclear  membrane 
and  is  connected  with  an  end  of  the  spireme  (figs.  51-54).  At  first  it 
is  quite  small,  and  it  gradually  increases  in  size  during  the  spireme 
stage.  There  is  no  "  bouquet  stage  "  in  this  form.  Figure  55  shows 
the  spireme  segmented  and  split  longitudinally.  The  segments  have 
begun  to  open  out  at  the  center  to  give  the  cross  which  is  the  typical 
tetrad  form  in  Stenopelmatus.  Figures  56,  58,  59,  and  60  show  various 
stages  in  the  contraction  of  the  split  segments  to  form  crosses  and 


6  STUDIES    IN    SPERMATOGENESIS. 

diamond-shaped  rings.  The  tetrads  usually  remain  connected  by 
delicate  linin  threads,  as  shown  in  figures  57  and  60,  also  in  figures 
62  and  63,  the  latter  taken  from  the  metaphase  of  the  first  maturation 
spindle.  If  these  linin  connections  persist,  as  they  appear  to  do,  from 
the  segmentation  of  the  spireme  to  metakinesis,  the  first  division  of 
the  contracted  tetrads  must  be  longitudinal,  corresponding  to  the  split 
in  the  segments  of  figures  55,  57,  58,  etc.  The  chromosomes  in  the 
metaphase  usually  appear  as  dumbbells  (fig.  66)  or  elongated  crosses 
(fig.  67),  but  occasionally  one  can  be  found  which  still  shows  its 
tetrad  nature  (fig.  64),  so  clearly  indicated  in  the  quadrivalent  crosses 
of  figure  59.  In  the  anaphase  the  chromosomes  are  often  split  as  in 
figure  68,  and  occasionally  the  two  components  can  be  seen  as  plainly 
as  in  figure  65.  Figure  61  shows  the  various  shapes  assumed  by  the 
element  x  during  the  tetrad-stage  of  the  chromosomes.  This  element 
x  almost  invariably  appears  in  a  vesicle  near  one  pole  of  the  spindle 
(figs.  67,  68);  in  exceptional  cases  it  is  found  nearer  the  equatorial 
plate,  as  in  figure  66,  or  even  in  the  same  plane  with  the  ordinary 
chromosomes,  but  always  somewhat  isolated  from  them.  In  position 
and  form  this  element  resembles  the  accessory  chromosomes  described 
by  Baumgartner  ('04)  for  Gryllus  domesticus  ;  in  its  mode  of  origin  it 
seems  to  differ  from  the  other  accessory  chromosomes  yet  described. 

Figures  69  and  70  show  the  23  bivalent  chromosomes  in  metaphase  ; 
in  figure  69  the  element  x  is  shown  partly  behind  the  large  chromo- 
some and  at  a  different  level.  In  figures  66  and  67  the  one  excep- 
tionally large  chromosome  doubtless  represents  the  two  larger  ones  of 
the  spermatogonia.  In  the  anaphase  the  element  x  is  sometimes  as 
conspicuous  as  in  figure  7 1  ;  in  other  cases  it  is  concealed  either  behind 
or  within  the  polar  mass  of  chromatin.  In  this  form  there  is  a  dis- 
tinct resting  stage  between  the  two  maturation  mitoses  (figs.  72-75). 
The  element  x  is  conspicuous  in  one-half  of  the  cells  (figs.  72,  73) ;  it 
may  be  included  in  the  nucleus  as  in  figure  72,  or  it  may  be  partly  or 
wholly  outside,  as  in  figures  74,  75,  and  76.  In  the  latter  case,  but 
not  in  the  former,  it  is  surrounded  by  its  own  membrane.  As  the 
chromatin  begins  to  condense  for  the  second  mitosis,  disintegration 
of  the  element  x  becomes  apparent.  This  is  most  easily  made  out  in 
cases  where  the  element  is  isolated,  as  in  figures  75  and  76  ;  but  there 
seems  to  be  little  doubt  that  it  disappears  before  the  metaphase  of  the 
second  maturation  mitosis.  It  is  not  possible  to  count  the  chromosomes 
in  this  stage,  they  are  so  crowded  together,  but  it  is  not  probable  that 
such  a  conspicuous  chromatin  element  as  that  seen  in  the  first  division 
could  escape  detection,  even  if  it  were  in  the  equatorial  plate  among 
the  chromosomes.  No  aberrant  element  is  ever  seen  in  these  spindles ; 


STENOPELMATUS.  7 

and,  moreover,  all  of  the  spindles  and  all  of  the  spermatids  appear  to 
be  exactly  alike  at  the  same  stage.  The  chromosomes  are  double  in 
the  prophase  (fig.  77)  and  always  appear  double  in  the  equatorial  plate 
(fig.  78),  the  paired  elements  corresponding  to  those  of  figure  65. 

In  figure  80,  plate  in,  a  pair  of  spermatids  is  shown  with  nuclear 
membrane  formed  and  the  spindle  fibers  twisted  in  a  characteristic 
manner.  Figure  81  is  a  slightly  later  stage  with  the  spindle-remains 
massed  against  the  nuclear  membrane.  Curiously  enough  there 
appears  in  the  nucleus  of  every  spermatid  a  body  similar  to  the  element 
x  of  the  spermatocytes  of  the  first  order  (figs.  82-86).  This  body  is 
often  applied  to  the  nuclear  membrane  and  connected  with  the  spireme 
(figs.  84-86).  It  decreases  in  size  and  finally  disappears  (figs.  88-91). 
The  spindle-remains  divides  (fig.  83),  and  a  small  part  of  it  (a)  goes  to 
form  the  acrosome  at  the  apex  of  the  head  (figs.  85-92).  The  larger 
part  is  probably  utilized  in  the  formation  of  the  tail,  for  it  gradually 
disappears  as  the  tail  develops. 

The  centrosome  which,  although  small,  is  conspicuous  in  each 
mitosis,  is  seen  in  figure  83  between  the  two  parts  of  the  spindle- 
remains,  applied  to  the  outside  of  the  nuclear  membrane.  In  figures 
85,  86,  and  87  the  relation  of  the  tail  (or  its  axial  fiber)  to  the  centro- 
some is  shown.  In  figures  87  and  88,  instead  of  the  small  spherical 
centrosome  of  figures  83  to  86,  we  have  a  much  elongated  body,  at 
first  (fig.  87)  applied  for  its  whole  length  to  the  nuclear  membrane,  but 
later  lying  along  one  side  of  a  middle  piece  (m),  as  shown  in  figure  89, 
and  in  a  later  stage  in  figures  90  to  92.  The  mature  spermatozoon 
with  its  forked  anterior  end  appears  in  figure  93. 

The  points  of  especial  interest  in  the  spermatogenesis  of  Stenopel- 
matus  are  the  development  of  the  aberrant  chromatin  element  x  during 
the  growth  stage  of  the  spermatocyte  of  the  first  order,  its  distribution 
to  one-half  of  the  spermatocytes  of  the  first  order,  its  disappearance 
during  the  rest  stage  between  the  two  maturation  divisions,  and 
the  development  of  a  similar,  though  smaller,  element  in  all  of  the 

spermatocytes. 

Blattella  germanica. 

Unlike  the  spermatogonia  of  Stenopelmatus ',  those  of  Blattella  have 
both  a  faintly-staining  nucleolus  and  a  deeply-staining  chromatin 
element  (x),  and  moreover  the  two  are  always  closely  associated  (figs. 
95,  96).  The  number  of  chromatin  elements  in  the  equatorial  plate 
of  late  spermatogonial  mitoses  is  23  (fig.  97).  Later  events  indicate 
that  one  of  the  23  is  the  element  x,  but  it  is  impossible  to  distinguish 
it  here.  Figure  98  is  a  very  early  stage  of  the  spermatocyte  of  the  first 
order,  showing  the  element  x  as  a  U-shaped  body.  The  centrosome 


8  STUDIES    IN    SPERMATOGENESIS. 

was  also  conspicuous  in  all  of  the  cells  of  this  group.  The  spireme 
here,  as  also  in  figure  99,  is  fine  and  closely  interwound.  In  figure 
99  and  again  in  figure  100  the  element  x  is  joined  to  the  spireme  as  it 
is  throughout  the  spireme  stage.  In  the  ' '  bouquet  "  or  "  polarized  ' ' 
stage  the  combined  nucleolus  and  element  x  are  always  at  one  side  of 
the  group  of  loops  and  down  very  close  to  the  base  of  the  figure  (figs. 
101,  103).  In  figure  102  most  of  the  loops  are  cut  across.  The  stage 
shown  in  figures  104  and  105  is  a  later  one  than  that  just  described. 
Here  we  have  again  a  continuous  spireme  connected  with  the  element 
x,  making  it  seem  improbable  that  the  bivalent  chromosomes  are 
really  separated  in  the  bouquet  stage.  Figure  106  gives  some  of  the 
variations  in  form  of  the  combined  nucleolus  and  element  x.  The  last 
of  the  five  figures  was  taken  from  a  giant  cell  containing  at  least  twice 
the  usual  amount  of  chro matin.  In  one  giant  cell  four  unusually 
large  combinations  of  this  kind  were  found,  and  a  corresponding 
amount  of  chromatin  in  the  spireme.  In  figure  107  one  sees  the 
spireme  divided  into  segments  still  joined  by  linin  bridges.  In  figure 
1 08  similar  segments  may  be  seen,  one  of  them  showing  a  longitudinal 
split.  The  element  x  is  present,  but  the  nucleolus  has  disappeared. 
In  many  cases  the  split,  if  it  appears  at  all,  closes  quickly  and  the 
chromosome  bends  in  U -shape,  as  in  figure  109,  plate  iv.  This  figure 
also  shows  two  centrosomes  (c).  In  other  cases  the  split  persists  as  in 
figure  1 10  and  leads  to  the  formation  of  crosses  of  a  tetrad  character 
(figs,  in,  112,  113),  as  in  Stenopelmatus  and  many  other  insects. 
Figures  114  to  117  show  later  stages  of  the  U-shaped  chromosomes. 
Perfect  rings  are  rare.  All  sorts  of  variations  are  seen,  broad  and 
narrow  U -shapes,  rings  split  at  one  point  or  the  opposite  points,  a 
U  split  at  the  bottom  (fig.  114),  pairs  of  parallel  rods  (fig.  115),  and 
occasionally  rods  constricted  in  the  middle  and  showing  a  longitudinal 
split  in  each  half,  as  in  figure  116.  Figure  117  shows  different  views 
of  the  split  rings.  Apparently  all  of  these  forms  straighten  out  so  that 
the  two  components  of  the  bivalent  chromosome  stand  end  to  end  as 
dumbbells  or  compressed  crosses  in  the  metaphase  of  the  first  matura- 
tion spindle  (figs.  123-125).  The  element  x  remains  concentrated  and 
more  or  less  spherical  in  form.  Figures  118-122  are  equatorial  plates, 
with  x  absent  in  figure  120,  in  the  same  plane  as  the  n  other  chromo- 
somes in  figure  119,  far  to  one  side  in  figure  118,  and  near  one  pole  of 
the  forming  spindle  in  figure  122.  It  is  also  shown  in  various  positions 
with  regard  to  the  spindle  in  figures  123  to  126  and  128  to  132.  In 
figure  125  it  is  apparently  double,  and  again  in  figure  129.  In  figure 
130  one  lagging  chromosome  shows  the  dyad  nature  of  the  products 


BLATTELLA   GERMANICA.  9 

of  the  division  of  the  tetrad.  In  this  form  there  can  be  no  doubt  that 
reduction  occurs  in  the  first  spertnatocyte  division.  The  element  x  is 
very  often  concealed  by  the  polar  aggregation  of  chromatin,  but  it  is 
sometimes  as  conspicuous  as  in  figures  131  and  132.  The  spermato- 
cytes  of  the  second  order  go  into  a  complete  resting  stage  before  they 
are  completely  separated,  and  one  of  a  pair  shows  the  element  x,  while 
it  is  lacking  in  the  other  (fig.  133).  At  the  close  of  the  resting  stage 
the  chromosomes  appear  as  1 1  pairs  of  rods  of  considerable  length, 
which  gradually  shorten  and  thicken  and  usually  bend  at  the  center, 
forming  U's  or  V's  (figs.  134-138).  In  one  stage  these  double  U's 
look  much  like  tetrads  (fig.  138).  The  rods  straighten  again  as  they 
shorten  still  more  (fig.  139),  become  more  closely  approximated,  and 
finally  form  dumbbells,  as  in  figure  141. 

The  element  x  is,  of  course,  present  in  only  one-half  of  these 
nuclei.  In  the  equatorial  plate,  figure  142,  it  is  absent ;  in  figure  143 
it  is  present,  but  can  not  be  distinguished  from  the  other  chromosomes, 
while  in  figure  144  it  is  rendered  conspicuous  by  its  spherical  form 
and  isolated  position.  In  only  a  few  cases  has  it  been  possible  to  dis- 
tinguish x  in  the  spindle.  Figures  146  and  147  show  two  of  these 
cases  where  this  element  is  clearly  double  and  of  different  form  from 
the  other  chromosomes.  It  is  probable  that  it  divides  and  so  goes 
into  one-half  of  all  of  the  spermatids,  as  in  McClung's  typical  cases 
of  the  accessory  chromosome.  Figure  145  shows  the  usual  appear- 
ance of  the  other  chromosomes  in  metaphase.  The  two  spermatids  of 
a  pair  are  always  alike  so  far  as  any  evidence  of  the  presence  of  the 
element  x  is  concerned  (fig.  148).  Figure  149  is  an  exceptional  case, 
where  one  chromatin  element  (possibly  x)  has  evidently  divided  late 
and  been  left  out  in  the  cytoplasm ;  a  smaller  chromatin  granule 
is  also  present  in  the  cytoplasm  of  each  spermatid.  All  of  the  sper- 
matids, as  in  Stenopelmatus ,  develop  a  deeply-staining  body,  which, 
however,  in  this  case  is  usually  centrally  located  and  often  appears 
double  (figs.  150-152). 

The  spindle-remains  (Spindelreste)  forms  a  very  conspicuous  body  at 
one  side  of  the  nucleus  in  the  spermatids,  and  occasionally  a  mass  of 
chromatin,  probably  due  to  imperfect  mitosis,  is  found  near  the  spindle- 
substance  (fig.  150).  The  mass  of  spindle-substance  at  first  appears 
structureless,  but  soon  assumes  the  condition  shown  in  figures  150  to 
152.  In  one  individual  many  of  the  spermatids  had  two  balls  of  spindle- 
material  (fig.  152),  and  the  resulting  later  stages  were  double-tailed 
(fig.  153).  Figure  156  shows  how  the  spindle-substance  goes  into  the 
tail  and  gradually  disappears  as  the  tail  lengthens. 


IO  STUDIES    IN    SPERMATOGENESIS. 

The  centrosome  is  evidently  applied  to  the  nuclear  membrane,  as  in 
Stenopelmatus ',  and  the  middle-piece  is  developed  in  connection  with 
it,  as  in  figures  156-157,  154-155,  158-160.  The  element*  of  the 
spermatids  gradually  disappears  (figs.  150, 159).  An  acrosome  develops 
at  the  anterior  end,  the  head  condenses  and  lengthens,  and  we  have  the 
ripe  spermatozoon  (fig.  161).  The  tail  is  very  long  and  is  shown  only 
in  part. 

Of  the  forms  studied,  Blattella  alone  has  many  degenerate  sperm- 
atozoa. Some  follicles  have  none,  others  a  number  varying  perhaps 
from  one-fourth  to  three-fourths  of  the  whole  number.  No  evidence 
of  degeneracy  was  detected  among  the  young  spermatids  up  to  the 
stage  shown  in  figures  154-155,  where  a  few  like  figure  162  were  found. 
Most  of  the  degenerate  forms  occur  among  the  nearly  ripe  spermatozoa 
or  in  the  sperm-ducts.  Such  are  shown  in  figures  163  to  168,  The 
chromatin  is  strangely  broken  up  into  irregular  clumps,  and  probably 
no  two  of  these  degenerate  sperm-heads  can  be  found  which  are  alike. 
The  tails  are  always  imperfect.  The  distribution  and  varying  num- 
bers of  these  degenerate  spermatozoa  make  it  impossible  to  interpret 
their  condition  as  due  to  the  absence  of  the  accessory  chromosome, 
as  Miss  Wallace  does  in  the  spider.  The  only  probable  explanation, 
it  seems  to  me,  is  imperfect  mitosis.  Cases  where  more  or  less  chro- 
matin is  left  behind  in  the  cytoplasm,  especially  in  the  first  spermato- 
cyte  mitosis,  are  very  common,  and  such  cases  as  those  shown  in 
figures  149  and  150  are  not  rare.  The  giant  cells,  so  far  as  I  have 
been  able  to  trace  them,  do  not  develop  into  spermatozoa. 

The  most  important  points  are  : 

(1)  The  presence  of  the  element  x  in  the  spermatogonia,  closely 
associated  with  the  nucleolus. 

(2)  The  uneven  number  of  chromatin  elements  in  the  metaphase  of 
spermatogonial  divisions. 

(3)  The  connection  of  the  element  x  with  the  spireme  up  to  the 
stage  where  the  spireme  segments  to  form  the  bivalent  chromosomes. 

(4)  The  varied  character  of  the  tetrads,  showing  the  first  spermato- 
cyte  division  to  be  a  reducing  division  in  the  sense  that  it  separates 
whole  chromosomes. 

(5)  The  fact  that  the  element  x  fails  to  divide  in  the  first  maturation 
division,  does  divide  in  the  second,  but  can  not  be  traced  beyond  the 
equatorial  plate  of  the  latter  mitosis. 

(6)  The  similarity  of  all  the  normal  spermatids,  though  one-half  of 
them  must  contain  the  element  x,  the  other  half  not. 

(7)  The  varying  and  often  large  number  of  degenerate  spermatozoa. 


BLATTELLA   GERMANICA.  II 

An  attempt  was  made  to  determine  the  somatic  number  of  chromo- 
somes. The  dividing  cells  of  the  follicles  of  young  eggs  seemed  to 
afford  the  most  favorable  material,  but  even  here  there  was  so  much 
overlapping  of  the  ends  of  the  chromosomes  that  it  was  impossible  to 
be  absolutely  certain  of  the  number.  In  the  two  most  favorable  cases 
23  were  counted  (fig.  94).  This  differs  from  McClung's  count  for 
similar  cases  among  the  Orthoptera,  and  Sutton's  for  Brachystola 
magnet.  The  eggs  have  so  far  resisted  all  efforts  to  learn  what  part 
the  odd  chromosome  may  play  in  fertilization. 

Tenebrio  molitor. 

In  the  metaphase  of  all  spermatogonial  mitoses  where  it  was  pos- 
sible to  count  accurately,  20  chromosomes  were  found,  19  large  ones 
of  approximately  equal  size,  and  i  small  spherical  one  (figs.  169,  170). 
There  is  nothing  in  the  resting  nucleus  of  the  spermatogonia  which 
would  suggest  either  a  nucleolus  or  an  accessory  chromosome.  The 
chromatin  stains  well  during  the  whole  growth  period  of  the  sperma- 
tocytes,  but  it  is  impossible  to  separate  the  period  into  so  definite 
stages  as  in  most  other  forms. 

In  the  youngest  spermatocytes  one  finds  occasionally  a  cyst  con- 
taining cells  with  nuclei  like  those  of  figures  171  and  172,  indicating 
that  a  brief  ' '  synapsis  ' '  or  condensation  stage  occurs  at  the  close  of 
the  last  spermatogonial  mitosis.  During  the  greater  part  of  the  period 
the  chromatin  forms  a  heavy,  irregular,  and  often  segmented  spireme 
(figs.  173,  174).  Shortly  before  the  first  maturation  division,  such 
split  segments  as  appear  in  figure  175  are  sometimes  found  ;  some  of 
these  simulate  tetrads  with  slender  connecting  bands  between  the 
paired  elements.  Again,  one  finds  a  few  cases  like  figure  176,  where 
the  spireme  is  segmented  into  bivalent  chromosomes,  each  component 
showing  a  longitudinal  split.  This  figure  also  shows  the  small  chro- 
mosome. Usually,  however,  the  irregular  and  much  tangled  spireme 
(figs.  173,  174)  condenses  into  a  heavy  segmented  band  variously  dis- 
posed in  the  nucleus  (fig.  177).  This  band  soon  separates  into  the 
bivalent  chromosomes  shown  in  figures  178  and  179,  giving  9  sym- 
metrical pairs  and  i  unsymmetrical  one  (fig.  179  s)  composed  of  the 
small  chromosome  and  a  much  larger  mate.  In  the  prophase  of  the 
spindle,  in  rare  cases,  some  of  the  chromosomes  are  longitudinally 
split  and  transversely  constricted,  forming  tetrads  (fig.  180),  but  more 
often  they  appear  as  in  figure  181.  The  unequal  pair  appears  in  each 
figure  at  s.  In  the  metaphase  (fig.  182)  it  is  the  last  to  come  into  the 
equatorial  plate,  possibly  because  of  its  lack  of  symmetry.  The  smaller 
component  of  this  pair  is  always  directed  toward  the  equator  of  the 


12  STUDIES    IN    SPERMATOGENESIS. 

spindle.  Figure  183  shows  a  small  tangential  section  of  a  spindle  in 
metaphase,  containing  the  unequal  pair  and  one  equal  pair.  In  figure 
184  a  polar  view  of  a  metaphase  is  shown,  the  unequal  pair,  which 
was  somewhat  below  the  others,  being  indicated  by  stippling.  Figures 
184  a  and  185  show  that  the  unequal  components  of  the  unsymmetrical 
pair,  as  well  as  the  equal  components  of  the  symmetrical  pairs,  are 
separated  in  metakinesis,  making  this  clearly  a  reduction  division. 
Two  polar  plates  are  shown  in  figures  186  and  187,  one  containing  10 
equal  elements,  the  other  9  equal  ones  and  i  small  one.  The  telophase 
is  shown  in  figure  188.  There  is  no  resting  stage,  but  the  new  spindle 
is  formed  from  the  remains  of  the  old  one,  and  the  spindle-shaped 
mass  of  chromatin  seen  in  figure  188  either  passes  into  the  center  of 
the  new  spindle  or  becomes  enveloped  by  it.  The  double  chromo- 
somes separate  as  in  figures  189  and  190.  Figure  190  shows  the  small 
dyad,  and  figure  189  an  aberrant  one  which  may  be  its  mate.  The 
spindle  in  both  divisions  is  peculiar  in  having  outside  of  the  spindle 
proper  a  dense  mass  of  fibers  which,  in  osmic  material,  stain  deeply 
with  iron  hsematoxylin.  These  fibers  are  shown  in  all  the  figures  from 
174  to  196.  Figures  191  and  192  are  equatorial  plates  of  the  two  kinds 
of  spermatocytes  of  the  second  order,  figure  191  showing  the  small 
chromosome.  An  early  anaphase  appears  in  figures  193  and  194, 
which  show  both  the  small  and  larger  chromosomes  in  metakinesis. 
Figure  195  is  a  later  anaphase  containing  the  divided  small  chro- 
mosome. In  figure  196  are  shown  the  two  polar  plates  of  a  spindle 
corresponding  to  that  of  figure  195,  and  in  figure  197  the  polar  plates 
of  a  spindle  in  which  10  equal  chromosomes  have  been  divided.  In 
Tenebrio  molitor  the  spermatids  are  therefore  certainly  of  two  distinct 
kinds,  so  far  as  the  chromatin  content  is  concerned. 

In  most  of  the  young  spermatids,  after  the  nuclear  membrane  has 
formed,  there  appears  an  isolated  chromatin  element,  which  corre- 
sponds fairly  well  to  the  large  or  to  the  small  component  of  the  unsym- 
metrical pair,  separated  in  the  first  mitosis  and  divided  in  the  second. 
The  clear  portion  of  the  nucleus  containing  this  isolated  element  is  at 
first  turned  toward  the  spindle-remains  (fig.  198),  but  before  the  tail 
appears  either  the  whole  nucleus  or  its  contents  have  rotated  180° 
(fig.  199).  Various  stages  in  the  development  of  the  spermatid  are 
seen  in  figures  200  to  203.  The  clear  region  and  the  isolated  element 
finally  disappear  (fig.  202  6),  and  the  chromatin  breaks  up  into  coarser 
and  then  into  finer  granules  within  the  sperm-head.  In  the  later  stages 
the  centrosome  is  clearly  seen  at  the  base  of  the  head  (fig.  203). 

In  order  to  determine,  if  possible,  the  value  of  the  unsymmetrical 
pair  of  chromatin  elements,  very  young  ovaries  and  ovaries  with  egg- 


TENEBRIO    MOLITOR.  13 

tubes  were  sectioned  and  the  chromosomes  counted  in  the  dividing 
cells  of  the  egg-follicle  (  <?  somatic  cells),  and  in  dividing  oogonia. 
In  both  cases  20  large  chromosomes  were  found.  Figure  207  is  the 
equatorial  plate  from  a  female  somatic  cell  of  a  young  egg-follicle. 
Figure  208  a  and  b  shows  two  sections  of  an  oogonium  in  the  prophase 
of  mitosis.  In  order  to  determine  the  number  and  character  of  the 
chromosomes  in  the  male  somatic  cells,  several  male  pupae  were  sec- 
tioned. As  in  the  spermatogonia,  19  large  chromosomes  and  i  small 
one  were  found.  Figure  204  shows  the  equatorial  plate  of  a  dividing 
male  somatic  cell,  and  figures  205  to  206  are  daughter  plates  from  a 
similar  cell.  (Three  large  chromosomes  of  the  plate  shown  in  figure 
206  are  in  another  section.) 

From  these  facts  it  appears  that  the  egg-pronucleus  must  in  all 
cases  contain  10  large  chromosomes,  while  the  spermatozoon  in  fertili- 
zation brings  into  the  egg  either  10  large  ones  or  9  large  ones  and  i 
small  one.  Since  the  somatic  cells  of  the  female  contain  20  large 
chromosomes,  while  those  of  the  male  contain  19  large  ones  and  i 
small  one,  this  seems  to  be  a  clear  case  of  sex-determination,  not  by 
an  accessory  chromosome,  but  by  a  definite  difference  in  the  character 
of  the  elements  of  one  pair  of  chromosomes  of  the  spermatocytes  of  the 
first  order,  the  spermatozoa  which  contain  the  small  chromosome 
determining  the  male  sex,  while  those  that  contain  10  chromosomes 
of  equal  size  determine  the  female  sex.  This  result  suggests  that  there 
may  be  in  many  cases  some  intrinsic  difference  affecting  sex,  in  the 
character  of  the  chromatin  of  one-half  of  the  spermatozoa,  though  it 
may  not  usually  be  indicated  by  such  an  external  difference  in  form  or 
size  of  the  chromosomes  as  in  Tenebrio.  It  is  important  that  related 
forms  should  be  studied  in  order  to  ascertain  whether  the  same  chro- 
matic conditions  prevail  in  other  species  of  this  genus  or  possibly  in 
the  Coleoptera  in  general.* 

Aphis  oenotherae. 

The  spermatogensis  of  Aphis  has  been  fully  described  in  another 
paper  and  will  merely  be  briefly  summarized  here  for  the  purpose  of 
comparison  with  other  forms. 

The  spermatogonia  contain  a  large  nucleolus,  which  gradually 
disappears  in  the  prophases  of  mitosis  (plate  vn,  figs.  209-211).  The 
youngest  spermatocytes  closely  resemble  the  spermatogonia  (fig.  212). 
There  is  no  bouquet  stage  and  no  such  marked  spireme  stage  as  in 


*  Prof.  E.  B.  Wilson  has  recently  found  a  similar  dimorphism  in  the  spermatozoa  of 
Lygceus  and  other  of  the  Hemiftera  heteroftera. 


14  STUDIES   IN    SPERMATOGENESIS. 

many  other  insects.  The  true  synapsis  occurs,  as  shown  in  figure 
213,  by  pairing  of  like  chromosomes  side  by  side.  This  conjugation 
of  like  chromosomes  is  followed  by  a  stage  in  which  they  are  massed 
together  at  one  side  of  the  nucleus  (fig.  214).  In  these  latter  stages 
the  nucleolus  has  entirely  faded  out  and  nothing  suggesting  an  acces- 
sory chromosome  is  present.  Figures  215  and  216  are  equatorial 
plates  of  the  first  spermatocyte  mitosis.  There  are  5  chromosomes  of 
different  sizes  and  shapes,  and  figure  216  shows  each  one  double. 
The  first  division  of  the  chromosomes,  though  apparently  longitudinal, 
is  evidently  a  separation  of  the  elements  paired  in  a  preceding  stage, 
and  is  therefore  a  reducing  division . 

The  anaphase  of  the  same  mitosis  is  shown  in  figures  217  and  218  ; 
it  is  peculiar  in  that  one  chromosome  always  divides  more  slowly  than 
the  others,  the  two  elements  hanging  together  at  one  end.  In  figure 
219  are  sister  spermatocytes  of  the  second  order,  the  "  lagging  "  chro- 
mosomes still  connected.  The  second  maturation  division  is  seen  in 
metaphase  in  figure  220  and  in  anaphase  in  figure  221.  Figure  222 
shows  a  young  spermatid,  the  five  chromosomes  still  preserving  their 
characteristic  form.  Figure  223  is  the  equatorial  plate  of  the  first 
maturation  division  of  the  winter  egg,  showing  the  same  form  and  size 
relations  of  the  chromosomes  as  in  the  spermatocyte  divisions.  Figures 
224  and  225  are  equatorial  plates  of  a  polar  spindle  (fig.  224)  and  of 
a  segmentation  spindle  (fig.  225)  of  the  parthenogenetic  egg,  where  10 
chromosomes  are  present,  2  of  each  of  the  sizes  found  in  the  sexual 
germ  cells. 

So  far  as  an  accessory  chromosome  or  any  other  visible  evidence 
of  a  sex  determinant  are  concerned,  the  results  are  entirely  negative. 
The  conditions  shown  do,  however,  support  Mendel's  conception  of 
the  "  purity  of  the  germ-cells,"  and  also  afford  evidence  in  favor  of 
Boveri's  theory  of  the  individuality  of  the  chromosomes. 

Sagitta  bipunctata. 

In  connection  with  these  insect  forms  it  is  of  interest  to  find  in  the 
sperm  atogenesis  of  Sagitta  a  body  which  stains  like  chromatin  and 
behaves  somewhat  like  the  accessory  chromosome.  It  is  found  in  all 
resting  stages  of  the  spermatogonia,  closely  applied  to  the  nuclear 
membrane  (fig.  226).  It  divides  before  each  spermatogonial  mitosis 
(fig.  227)  and,  though  not  often  discernible  in  the  spindle,  appears  in 
the  next  generation.  Figure  228  is  the  last  spermatogonial  mitosis, 
and  figure  229  shows  the  element  x,  and  the  chromosomes  paired  at 
one  pole  of  the  spindle.  During  the  various  phases  of  the  growth 
stage  (figs.  230-232)  the  element  x  is  again  applied  to  the  nuclear  mem- 


SAGITTA   BIPUNCTATA.  15 

brane.  In  the  prophase  of  the  first  maturation  division  this  element 
divides  (figs.  233-234),  and  in  metakinesis  the  two  elements  are  found 
in  various  positions  with  regard  to  the  spindle  (figs.  235-237),  often  as 
conspicuous  as  in  these  figures,  but  sometimes  concealed  among  the 
chromosomes.  Before  the  spindle  for  the  second  division  forms,  this 
element  divides  again  and  one  of  the  products  goes  into  each  spermatid 
(figs.  238-241). 

As  Sagitta  is  hermaphrodite,  there  would  appear  to  be  no  question 
of  sex  determination  by  any  special  chromatic  element.  The  size  of 
the  element  x,  its  evident  chromatic  nature,  its  division  before  each 
mitosis,  and  its  presence  in  mitosis  and  in  the  spermatids,  with  the  same 
staining  qualities  as  in  the  previous  rest  stages,  certainly  indicate 
some  important  function,  either  in  the  whole  process  of  spermato- 
genesis  or  in  the  formation  of  the  sperm-head,  of  which  it  finally 
becomes  a  part.  In  Sagitta  this  element  certainly  can  not  be  regarded 
as  a  specialized  spermatogonial  chromosome,  or  as  chromatin  rejected 
from  the  spireme.  No  such  element  is  present  in  the  ovogenesis  of 
Sagitta  (Stevens,  '03),  nor  has  any  been  detected  in  connection  with 
fertilization.  It  is  certain  that  none  is  present  in  the  first  segmenta- 
tion spindle  of  the  egg. 


GENERAL  DISCUSSION. 
THE  "ACCESSORY  CHROMOSOME." 

The  literature  bearing  on  the  ' '  accessory  chromosome ' '  of  McClung, 
the  ' '  small  chromosomes ' '  of  Paultnier,  and  the  ' '  chromatin  nucleoli ' ' 
of  Montgomery  has  been  fully  discussed  by  McClung  in  the  paper 
entitled,  "The  accessory  chromosome — sex  determinant?  "  ('02),  and 
will  therefore  be  considered  here  only  in  its  relation  to  the  several 
forms  studied.  The  present  status  of  the  question  has  been  well  sum- 
marized more  recently  by  Montgomery  under  the  heading  ' '  Hetero- 
chromosomes  ' '  in  the  paper,  ' '  Some  observations  and  considerations 
upon  the  maturation  phenomena  of  the  germ  cells." 

Three  theories  as  to  the  function  of  the  ' '  heterochromosomes ' ' 
have  been  advanced  :  (i)  That  of  McClung  that  they  are  sex-deter- 
minants, since  in  the  forms  which  he  has  examined  these  chromatin 
bodies  occur  in  only  one-half  of  the  spermatozoa,  and  the  sex-char- 
acter is  the  only  character  which  divides  the  individuals  of  a  species 
into  two  approximately  equal  groups.  (2)  That  of  Paulmier  and 
Montgomery  that  they  are  degenerating  chromatin.  Montgomery 
regards  them  as  ' '  chromosomes  that  are  in  the  process  of  disappear- 


1 6  STUDIES    IN    SPERM ATOGENESIS. 

ance  in  the  evolution  of  a  higher  to  a  lower  chromosome  number." 
(3)  That  of  Miss  Wallace,  who  suggests  that  in  the  spider  only  the 
one  out  of  each  four  spermatids  which  contains  the  accessory  chro- 
mosome is  capable  of  developing  into  a  functional  spermatozoon,  while 
the  other  three  degenerate,  as  do  the  polar  bodies  given  off  by  the  egg. 
McClung  is  inclined  to  believe  that  the  accessory  chromosome  is  an 
element  common  to  all  of  the  male  reproductive  cells  of  Arthropods, 
and  probably  to  vertebrate  spermatocytes  as  well  ('02). 

Of  the  insects  considered  in  this  paper  Aphis  and  Termopsis  have 
no  "accessory  chromosome  "  or  "  heterochromosome  "  of  any  kind. 
The  fact  that  no  males  develop  from  the  fertilized  eggs  of  Aphis  might 
be  offered  as  a  reason  for  its  absence,  but  such  an  argument  would 
not  apply  to  Termopsis.  The  sex-character  may  indeed  be  repre- 
sented in  the  chromatin  of  some  one  of  the  pairs  of  paternal  and 
maternal  chromosomes  of  the  spermatocytes,  but  there  is  no  evident 
peculiarity  by  which  one-half  of  the  spermatozoa  can  be  said  to  be 
different  from  the  other  half.  As  to  McClung's  statement  ('02)  ' '  that 
if  there  is  a  cross-division  of  the  chromosomes  in  the  maturation 
mitosis,  there  must  be  two  kinds  of  spermatozoa,  regardless  of  the 
presence  of  the  accessory  chromosome,"  it  appears  to  me  that  in  a 
case  like  the  aphid,  where  the  paired  elements  of  the  five  bivalent 
chromosomes  are  separated  in  the  first  maturation  mitosis,  there  may 
be  as  many  as  seventeen  kinds  of  spermatozoa  instead  of  two.  If, 
however,  we  suppose  that  the  sex  characters  are  segregated  in  the 
first  maturation  mitosis,  there  would,  of  course,  be  two  equal  classes 
of  spermatozoa  with  reference  to  that  character. 

In  Stenopelmatus  the  element  x  in  certain  stages  closely  resembles 
the  "  accessory  chromosome  "  of  McClung,  and  especially  that 
described  by  Baumgartner  for  Gryllus  domesticus,  but  its  origin  and 
fate  are  different.  It  first  appears  attached  to  an  end  of  the  spireme 
in  the  growth  stage  of  the  young  spermatocytes,  where  it  is  much 
smaller  than  in  later  growth  stages.  It  gradually  increases  in  size,  is 
a  conspicuous  element  in  the  first  maturation  spindle,  goes  into  one 
of  each  pair  of  spermatocytes  of  the  second  order,  and  there  degenerates 
during  the  rest  stage  between  the  two  maturation  mitoses.  The  whole 
history  of  this  element  suggests  that  it  may  be  rejected  chromatin 
analogous  to  that  observed  in  the  ovogenesis  of  many  forms.  In 
Sagitta,  for  example,  a  considerable  quantity  of  chromatin  granules  is 
given  off  by  the  chromosomes  and  cast  out  into  the  cytoplasm  near 
the  close  of  ovogenesis  (Stevens,  '03).  Riickert  ('92)  has  described  a 
similar  casting  out  of  chromatin  material  by  the  chromosomes  of  the 
o  ocy tes  of  Pristiurus. 


GENERAL    DISCUSSION.  17 

The  spermatogeuesis  of  Stenopelmatus,  therefore,  differs  from  that 
of  the  other  Orthoptera  which  have  been  described  in  having  (i)  a 
larger  number  of  chromosomes  (46),  (2)  an  even  number  in  the  sper- 
matogonia,  (3)  an  accessory  chromatin  structure  in  the  spermatocytes 
of  the  first  order,  which  disappears  before  the  second  maturation 
division. 

\nBlattellavfe.  have  a  typical  "accessory  chromosome,"  accord- 
ing to  McClung.  It  appears  (i)  in  all  resting  spermatogonia  closely 
associated  with  a  nucleolus,  (2)  in  the  spermatogonial  mitoses  as  an 
odd  chromatin  element,  making  23  in  all,  (3)  in  the  growth  stage  of 
the  spermatocytes  connected  with  an  end  of  the  spireme  and  also  with 
the  nucleolus.  It  becomes  separated  from  the  other  chromatin  in  the 
tetrad-stage,  remains  nucleolus-like  in  form,  and  later  appears  in  the 
first  maturation  division  either  among  the  chromosomes  or  in  a  more 
or  less  aberrant  position.  It  passes  into  one  of  each  pair  of  sperma- 
tocytes of  the  second  order,  persists  during  the  rest  stage,  appears  in 
the  second  mitosis  as  a  dyad  and  then  divides,  going  into  one-half  of 
the  spermatids.  The  spermatids,  however,  as  in  Stenopelmatus,  all 
have  the  same  appearance  :  each  has  in  the  center — not  against  the 
nuclear  membrane — a  small  element  that  stains  like  chromatin.  Occa- 
sionally a  mass  of  chromatin  is  found  outside  the  nucleus,  but  this  is 
not  constant  enough  to  support  the  contention  of  Moore  and  Robinson 
('05)  that  the  ' '  nucleolus  "  of  the  related  form,  Periplaneta  americana, 
is  fragmented  and  cast  out  into  the  cytoplasm.  The  spermatids  all 
appear  to  develop  equally  well  for  some  time,  but  as  they  approach 
maturity  a  varying  proportion  of  them  become  degenerate.  This  can 
not,  however,  be  due  to  absence  of  the  accessory  chromosome,  as  Miss 
Wallace  supposes,  in  the  spider ;  for  in  some  follicles  no  degenerate 
spermatozoa  are  found,  and  in  others  more  than  half  may  be  degen- 
erate. All  attempts  to  study  fertilization  stages  of  the  egg  have  so 
far  failed,  and  the  chromosomes  in  the  female  somatic  cells  have  not 
proved  favorable  for  counting.  Twenty-three  have  been  counted  in 
several  cases,  but  there  was  always  some  chance  of  error.  If  23  is 
the  somatic  number  in  both  sexes,  it  must  be  maintained  by  union  of 
sex-cells  containing  n  and  12  chromosomes,  respectively,  the  same 
unequal  number  occurring  in  the  maturated  eggs  as  in  the  sperm. 
Under  such  conditions  it  is  difficult  to  see  how  the  odd  chromatin 
element  of  the  spermatozoa  can  determine  sex. 

The  brief  description  of  the  chromatin  element  x  in  Sagitta,  intro- 
duced here  because  it  behaves  like  the  accessory  chromosome  in 
many  particulars,  serves  as  an  example  of  the  occurrence  of  such  an 
element  in  the  spermatogenesis  of  a  hermaphrodite  form,  where  it  can 


l8  STUDIES    IN    SPERMATOGENESIS. 

not  possibly  be  conceived  of  as  a  sex  determinant.  In  Sagitta  it  is 
known  to  be  confined  to  the  male  germ-cells.  No  such  element 
occurs  in  the  ovogenesis,  in  the  sperm  nucleus  in  the  egg,  or  in  the 
first  segmentation  spindle.  Its  function  must,  therefore,  be  confined 
to  the  process  of  spermatogenesis. 

From  the  standpoint  of  sex  determination,  we  have  in  Tenebrio  moli- 
tor  the  most  interesting  of  the  forms  considered  in  this  paper.  In  both 
somatic  and  germ  cells  of  the  two  sexes  there  is  a  difference  not  in  the 
number  of  chromatin  elements,  but  in  the  size  of  one,  which  is  very 
small  in  the  male  and  of  the  same  size  as  the  other  19  in  the  female. 
The  egg  nuclei  of  the  female  must  be  alike  so  far  as  number  and  size 
of  chromosomes  are  concerned,  while  it  is  absolutely  certain  that  the 
spermatids  are  of  two  equal  classes  as  to  chromatin  content  of  the 
nucleus — one-half  of  them  have  9  large  chromosomes  and  i  small  one, 
while  the  other  half  have  10  large  ones.  Since  the  male  somatic  cells 
have  19  large  and  i  small  chromosome,  while  the  female  somatic  cells 
have  20  large  ones,  it  seems  certain  that  an  egg  fertilized  by  a  sperm- 
atozoon which  contains  the  small  chromosome  must  produce  a  male, 
while  one  fertilized  by  a  spermatozoon  containing  10  chromosomes  of 
equal  size  must  produce  a  female.  The  small  chromosome  itself  may 
not  be  a  sex  determinant,  but  the  conditions  in  Tenebrio  indicate  that 
sex  may  in  some  cases  be  determined  by  a  difference  in  the  amount  or 
quality  of  the  chromatin  in  different  spermatozoa.  This  is  much  the 
most  suggestive  part  of  the  work,  and  it  will  be  followed  up  by  the 
study  of  related  forms. 

There  appears  to  be  so  little  uniformity  as  to  the  presence  of  the 
heterochromosomes,  even  in  insects,  and  in  their  behavior  when  pres- 
ent, that  further  discussion  of  their  probable  function  must  be  deferred 
until  the  spermatogenesis  of  many  more  forms  has  been  carefully 
worked  out. 

BRYN  MAWR  COU^GE,  May  75,  1905. 


BIBLIOGRAPHY.  19 

BIBLIOGRAPHY. 

BAUMGARTNER,  W.  J. 

'04.  Some  new  evidences  for  the  individuality  of  the  chromosomes.    Biol.  Bull., 
vol.  8,  no.  i 

McCLUNG.  C.  E. 

'99.  A  peculiar  nuclear  element  in  the  male  reproductive  cells  of  insects.     Zool. 

Bull.,  vol.  2. 

'00.  The  spermatocyte  divisions  of  the  Acrididae.  Kans.  Univ.  Quart.,  vol.  9,  no.  i. 
'01.  Notes  on  the  accessory  chromosomes.     Anat.  Anz.,  bd.  20,  nos.  8  and  9. 
'02.  The  accessory  chromosome — Sex-determinant?  Biol.  Bull.,  vol.  3,  nos.  i  and  2. 
'02 a.  The  spermatocyte  divisions  of  the  Locustidae.     Kans.  Univ.  Quart.,  vol.  i, 

no.  8. 
MONTGOMERY,  THOS.  H.,  JR. 

'01.  A  study  of  the  chromosomes  of  the  germ-cells  of  Metazoa.     Trans.  Amer. 

Phil.  Soc.,  vol.  20. 
'01  a.  Further  studies  on  the  chromosomes  of  the  Hemiptera  heteroptera.     Proc. 

Acad.  Nat.  Sci.  Phila.  1901. 
04.  Some  observations  and  considerations  upon  the  maturation  phenomena  of  the 

germ-cells.     Biol.  Bull.,  vol.  6,  no.  3, 
MOORE,  J.  E.  S.,  and  ROBINSON,  L.  E. 

'05.  On  the  behavior  of  the  nucleolus  in  the  spermatogenesis  of  Periplaneta 

americana.     Quart.  Jour,  of  Mikr.  Sci.,  n.  s.,  no.  192  (vol.  48,  part  4). 
PAULMIER,  F.  C. 

'93.  Chromatin  reduction  in  the  Hemiptera.     Anat.  Anz.,  vol.  14. 
'99.  The  spermatogenesis  of  Anasa  tristis.     Journ.  of  Morph.,  vol.  15. 
RUCKERT,  J. 

'92.  Zur.  Entwickelungsgeschichte    des  Ovarialeies  bei  Selachiern.     Anat.  Anz., 

vol.  7,  no.  4  and  5. 
DE  SINETY,  R. 

'01.   Recherches  sur  la  biologie  et  1'anatomie  des  phasms.     La  Cellule,  vol.  191 
STEVENS,  N.  M. 

'03.  On  the  ovogenesis  and  spermatogenesis  of  Sagitta  bipunctata.    Zool.  Jahrb., 

vol.  18. 
SUTTON,  W.  S. 

'02.   On  the  morphology  of  the  chromosome  group  in  Brachystola  magna,     Biol. 

Bull.,  vol.  4,  no.  i. 

'03.   The  Chromosomes  in  heredity.     Biol.  Bull.,  vol.  4,  no.  5. 
WALLACE,  L.  B. 

'00.  The  accessory  chromosome  in  the  spider.     Anat.  Anz. ,  vol.  18. 
'05.  The  spermatogenesis  of  the  spider.     Biol.  Bull.,  vol.  8,  no.  3. 
WILCOX,  E.  V. 

"95.  Spermatogenesis  of  Caloptenus  femur-rubrum  and  Cicada  tibicen.     Bull. 

Mus.  Comp.  Zool.  Harvard  Univ.,  vol.  27. 
'96.   Further  studies  on  the  spermatogenesis  of  Caloptenus  femur-rubrum.     Bull. 

Mus.  Comp.  Zool.  Harvard  Univ.,  vol.  29. 
'97.  Chromatic  tetrads.     Anat.  Anz.,  vol.  14. 

'01.  Longitudinal  and  transverse  division  of  chromosomes.     Anat.  Anz.,  vol.  igj 
no.  13. 


2O  DESCRIPTION    OF    PLATES. 


[The  figures  of  plates  i-vi  were  all  drawn  with  Zeiss  oil-immersion  2  mm., 
oc.  12,  and  have  been  reduced  one-third ;  those  of  plate  vn  with  oc.  8,  not  reduced.] 

PLATE  I. 
Termopsis  angusticollis. 

FIGS.  1-3.  Resting  nuclei  of  spermatogonia,  showing  division  of  nucleolus. 

4.  Equatorial  plate  of  spermatogonial  mitosis,  52  chromosomes. 
5-6.  Young  spermatocytes,  showing  division  of  nucleolus. 

7.  First  maturation  spindle,  and  two  nuclei  (6  and  8)  in  same  cyst. 
8-10.  Skein-stage — so-called  synapsis-stage. 
11-14.  Bouquet-stage,  showing  two  nucleoli,  centrosome   (c)   in  fig.  n,  and 

loops  made  up  of  fine,  then  coarser  granules. 

15-17.  Stage  following  preceding;   loops  straightened  out  and  extending  in 
various  directions  through  nucleus. 

18.  a,  Chromosomes  much  shortened  and  longitudinally  split;  b,  chromo- 

somes contracted  to  form  diamond-shaped  figures. 

19.  Stage  between  180  and  i8&. 

20.  Stage  between  19  and  i8&. 

21.  Stage  similar  to  180,  one  chromosome  in  double  diamond  form. 

22.  First  maturation  spindle   in  metaphase,   chromosomes   in   single  and 

double  diamond  shapes. 

23.  Chromosome  in  single  diamond  or  tetrad  form,  as  they  usually  come 

into  the  spindle. 

24.  Double  diamond-form  assumed  before  metakinesis. 

25.  The  26  chromosomes  of  an  early  metaphase. 

26.  First  maturation  spindle  in  metakinesis. 

27.  Equatorial  plate  of  first  maturation  spindle  in  metakinesis. 

28.  Another  spindle,  showing  three  granules  which  are  probably  remains 

of  nucleoli. 

29.  Anaphase  of  first  maturation  mitosis,  one  centrosome  divided. 

30.  Late  anaphase. 

31-32.  Telophase,  exceptional  cases  of  division  of  the  cell. 

33-36.  Partial  rest  stage  between  first  and  second  maturation  divisions,  two 

nucleoli  present.    Chromosomes  in  fig.  36  in  form  of  double  diamonds 

ready  for  metakinesis. 
37-38.  Second  maturation  spindle  in  metaphase. 

39.  Equatorial  plate  of  second  maturation  spindle,  26  chromosomes. 

40.  Same  in  anaphase. 

41.  Four  spermatid  nuclei  in  one  cell,  each  nucleus  containing  one  nucleolus. 

42.  A  later  stage,  showing  elongation  of  nuclei,  centrosome  and  sphere  at 

posterior  end. 

43-45.  Later  stages  in  the  development  of  the  spermatozoa,  nucleolus  grows 
gradually  smaller. 


STEVENS. 


PLATE    I. 


'ft 


F 


Wit 


uu 


3* 


j 


© 

39 


TERMOPSIS    ANGUSTICOLLIS. 


22  DESCRIPTION    OF    PLATES. 


PLATE  II. 
Stenopelntatus. 

FIGS.  46-47.  Nuclei  of  spermatogonia,  showing  2  and  3  nucleoli  (M). 

48-49.  Prophase  of  spermatogonial  mitosis,  showing  two  exceptionally  large 

chromosomes  of  equal  length. 

50.  Equatorial  plate  of  spermatogonial  mitosis,  46  chromosomes. 
51-54.  Spermatocytes  in  spireme  stage,  nucleus  containing  a  nucleolus  (n), 
and  a  chromatin  element   (.*•),  which  is  attached  to  one  end  of 
spireme  and  gradually  increases  in  size  during  growth  stage  of 
Spermatocytes. 

55.  Spireme  longitudinally  split  and  showing  the  beginning  of  cross  for- 

mation. 

56.  Spireme  segmented,  tetrads  forming. 

57.  One  split  segment  and  a  part  of  another  connected  by  bands  of  linin. 

58.  More  open  cross  and  diamond  forms ;  element  x  conspicuous. 
59-60.  More  contracted  cross  and  diamond-shaped  tetrads ;  linin  bands  shown 

in  60,  where  element  x  is  also  present. 

61.  Different  forms  assumed  by  element  x  during  tetrad  stage  (figs.  56-60) 
62-63.  Diamond-shaped  and  contracted  cross-shaped  tetrads  from  metaphase 
of  first  maturation  mitosis,  showing  linin  connections. 

64.  Diamond-shaped  tetrad  with   spindle-fibers  attached;   a-a,  probably 

halves  of  one  univalent  chromosome ;  b-b,  halves  of  the  other. 

65.  Dyad  from  anaphase  of  first  maturation  mitosis. 

66-67.  Metaphase  of  first  maturation  spindle,  showing  element  x  in  different 

positions. 

68.  Late  anaphase  of  same. 

69-70.  Equatorial  plate  of  first  maturation  spindle,  23  chromosomes  and  ele- 
ment x  below,  in  fig.  69. 

71.  Chromatin  massed  at  poles  of  spindle;  element  x  isolated  at  one  pole. 
72-73.  Two  resting  Spermatocytes  of  the  second  order,  one  containing  ele- 
ment x,  the  other  not 
74-76.  Successive  stages  of  breaking  down  of  element  x. 

77.  Prophase  of  second  division;  dyads  evident,  but  no  sign  of  x  in  this 

or  following  stages. 

78.  Second  spermatocyte  division — metakinesis. 

79.  Same ;  late  anaphase. 


STEVENS. 


PLATE    II. 


N.  M.  S.  del. 


STENOPELMATUS. 


24  DESCRIPTION    OF    PLATES. 


PLATE  III.     • 
Stenopelmatus. 

FIG.   80.  Telophase  of  second  maturation  mitosis. 

81.  Young  spermatid,  showing  spindle-remains  at  .y. 

82.  Spermatid  showing  a  conspicuous  chromatin  element  in  nucleus,  and 

spindle-remains  (s)  elongated. 

83.  Spermatid,    showing    centrosome     (c)     and    divided    spindle-remains 

(s  and  a). 

84.  Older  spermatid,  showing  centrosome  (c),  axial  fiber  of  tail,  and  spindle- 

remains  CT). 

85.  Spermatid,  showing  acrosome  material  (a)  migrating  to  side  of  nucleus 

opposite  centrosome. 

86.  Slightly  older  spermatid. 

87.  Later  stage  of  spermatid,  showing  condensed  chromatin,  elongated  cen- 

trosome (c),  acrosome  material  (a),  and  spindle-remains  (s). 
88-89.  Older  spermatids,  showing  formation  of  acrosome  (a)  and  middle  piece(m). 
90-92.  More  advanced  stages. 
93.  Mature  spermatozoon. 

Blattella  germanica. 

FIG.   94.  Somatic  cell  from  egg  follicle,  23  chromosomes. 

95.  .Spermatogonium,   showing  chromatin  element    (.*•)    associated  with  a 

nucleolus  (w). 

96.  Same,  prophase  of  mitosis. 

97.  Equatorial  plate  of  spermatogonial  mitosis,  23  chromatin  elements. 

98.  Young  spermatocyte,  showing  centrosome  (c)  and  U-shaped  element  (x). 

99.  Young  spermatocyte,  element  x  attached  to  one  end  of  a  long,  fine  spi- 

reme. 

100.  Coarser  spireme  stage. 
101-103.  Bouquet  stage. 
104-105.  Later  spireme  stage. 

106.  Various  forms  assumed  by  the  combined  nucleolus  and  element  x;  last 

figure  from  a  giant  cell. 

107.  Segmenting  spireme. 

108.  Similar  stage  to  fig.  107,  one  chromosome  longitudinally  split;  element  x 

present. 


STEVENS. 


PLATE    III. 


HBP    I 

\/ 

106 


108 


26  DESCRIPTION    OF   PLATES. 


P^ATE  IV. 
Blattella  germanica. 

FIG.  109.  Similar  stage  to  figs.  107  and  108;  chromosomes  U-shaped  and  not  longi- 
tudinally split;  two  centrosomes  present  (c). 
no.  Longitudinally  split  chromosomes. 

111-113.  Various  stages  in  formation  of  cross-shaped  tetrads. 
114-117.  Bent  rods,  U-shapes,  split  rings,  pairs  of  rods,  and  rod-shaped  tetrads 

(116),  which  are  equivalent  to  the  crosses  of  figs.  112-113. 
118-122.  Metaphase  of  first  maturation  division,  showing  the  element  x  in  various 

positions. 
123-127.  First  maturation  spindle  in  metaphase. 

128.  Same  in  anaphase. 
129-132.  Late  anaphase,  showing  element  x  double  in  129,  and  a  lagging  tetrad 

in  130. 

133.  Telophase,  with  the  element  x  in  one  daughter  cell. 
134-136.  Prophase  of  second  maturation  mitosis,  showing  dyads  and  element  x. 


STEVENS. 


PLATE    IV. 


rr8 


It-!  -W 


130 


128 


-x          * 


135 


136 


N.  M.  8.  del. 


BLATTELLA    GERMANICA. 


28  DESCRIPTION    OF    PLATES. 


PLATE  V. 
Blattella  germanica. 

FIGS.  137-141.  Dyads  contracting  for  second  maturation  mitosis. 

142.  Equatorial  plate  of  second  maturation  spindle,  containing  II  chro- 
mosomes. 

143-144.  Same,  with  n  chromosomes  and  the  element  x. 
145-147.  Sections  of  second  maturation  spindles;  element  x  dividing  in  146 
and  147. 

148.  Telophase  of  second  mitosis. 

149.  Telophase  of  second  mitosis,  showing  masses  of  chromatin  left 

behind  in  cytoplasm. 

150.  Spermatid  with  extranuclear  chromatin  (a). 

151.  Similar  stage;  different  view  of  spindle-remains  (s~)  and  of  chro- 

matin element  (xi). 
152-153.  Spermatid  with  divided  spindle-substance  and  the  corresponding 

double-tailed  form. 
154-155-  Stages  between  156  and  158. 
156-157.  Older  spermatids  than  151,  showing  spindle-remains  (s)  and  cen- 

trosome  (c). 
158-160.  Later  stages  in  development  of  sperm-head. 

161.  Ripe  spermatozoon. 
162-168.  Degenerate  spermatids  and  spermatozoa. 


STEVENS. 


PLATE    V. 


'37 


'J* 


146 


168 


BLATTELLA    GERMANICA. 


3<3  DESCRIPTION    OF   PLATES. 

PX.ATB  VI. 
Tenebrio  molitor. 

FIGS.  169-70.  Equatorial  plates  of  spermatogonial  mitosis,  showing  19  large  and 

i  small  chromosome. 

I7I-I75-  Condensation  stage,  bouquet  stage,  spireme  stage,  and  rather  rare 
tetrad  stage  of  young  spermatocyte. 

176.  Bivalent  chromosomes,  with  longitudinal  split;  small  chromosome 

shown  at  s. 

177.  Bivalent  chromosomes  condensed  into  a  close  spireme. 

178-179.  Bivalent  chromosomes  separating  for  mitosis.    The  unsymmetrical 
pair  shown  in  fig.  179. 

180.  Prophase  of  first  maturation  mitosis,  showing  the  unsymmetrical 

pair  and  the  tetrad  nature  of  the  symmetrical  pairs. 

181.  Prophase  of  same  mitosis,  showing  symmetrical  and  unsymmetrical 

pairs,  as  in  figs.  178  and  179. 

182.  Metaphase,  unsymmetrical  pair  out  of  the  equatorial  plane. 

183.  Tangential  section  of  a  spindle  in  metaphase,  showing  the  unsym- 

metrical pair  and  one  symmetrical  pair. 

184.  Equatorial  plate  of  same  mitosis,  10  chromosomes. 

1840.  Early  anaphase,  showing  separation  of  the  elements  of  the  unsym- 
metrical pair. 

185.  Later  anaphase. 

186.  Polar  plate,  showing  9  large  and  i  small  chromosome. 

187.  Polar  plate,  showing  10  large  chromosomes. 

188.  Condensation  stage  between  the  two  maturation  divisions. 
189-190.  Prophase  of  second  maturation  division,  fig.  189  showing  10  equal 

dyads,  and  fig.  190,  showing  9  equal  and  I  small  dyad. 

191.  Equatorial  plate,  showing  i  small  chromosome  and  9  large  ones. 

192.  Equatorial  plate,  showing  10  large  chromosomes. 
I93-I94-  Tangential  sections  of  spindle  in  metakinesis. 

195.  Anaphase  of  same  mitosis. 

196.  Polar  plates  of  a  spindle,  showing  in  each  i  small  chromosome  and 

9  large  ones. 

197.  Polar  plates  of  another  spindle,  10  large  chromosomes  in  each. 

198.  Young  spermatid,  showing  isolated  small  chromosome. 

199.  Young  spermatid,  showing  isolated  large  chromosome  and  rotation 

of  nuclear  contents. 
200-2020,  b.  Older  spermatids. 

203.  Sperm-heads,  showing  centrosome  and  granular  chromatin. 

204.  Equatorial  plate  from  dividing  somatic  cell  of  male  pupa,  showing 

19  large  and  I  small  chromosome. 

205-206.  Daughter  plates  of  a  similar  spindle,  showing  small  chromosome 
in  each ;  three  of  the  large  chromosomes  missing  in  206. 

207.  Equatorial  plate  of  a  dividing  cell  of  follicle  of  a  young  egg,  show- 

ing 20  large  chromosomes. 

208.  Prophase  of  mitosis  in  a  young  oogonium,  showing  20  large  chro- 

mosomes in  two  sections,  a  and  b. 


STEVENS. 


PLATE    VI. 


772 


175 


177 


178 


179 


^ 

••^-- 


iSi 


«M 


-     ..-.t,  . 

•A 


'83 


188 


196 


/97 


204 
N.  M.  8.  deL 


799 


205 


'94 


208  a 


•    ;  !  <'• 

W 

195 


203 


io8  b 


32  DESCRIPTION    OF    PLATES. 


PLATE  VII. 
Aphis  oenotherae. 

FIG.  209.  Spermatogonium. 
210-211.  Spermatogonia  in  prophase  of  mitosis. 

212.  Young  spermatocyte  of  first  order. 

213.  Spermatocytes  of  first  order;  conjugation  of  the  chromosomes. 

214.  Condensation  of  chromatin — spermatocytes  of  first  order  immedi- 

ately before  mitosis. 

215.  Equatorial  plate  of  first  maturation  division. 

216.  Same,  side  view,  showing  chromosomes  double. 
217-218.  Anaphase  of  same  mitosis. 

219.  Daughter  spermatocytes  of  second  order. 

220.  Equatorial  plate  of  second  maturation  mitosis. 

221.  Anaphase  of  same. 

222.  Young  spermatid. 

223.  Equatorial  plate  of  first  polar  spindle  of  winter  egg. 

224.  Equatorial  plate  of  polar  spindle  of  parthenogenetic  egg. 

225.  Equatorial  plate  of  segmentation  spindle  of  parthenogenetic  egg. 

Sagitta   bipunctata. 

FIG.  226.  Resting  Spermatogonia. 

227.  Prophase  of  spermatogonial  mitosis. 

228.  Last  spermatogonial  mitosis,  metakinesis. 

229.  Anaphase  of  same,  showing  synapsis  of  chromosomes  at  pole  of 

spindle,  and  element  x. 

230.  Resting  spermatocyte  of  first  order. 

231.  Bouquet  stage. 

232.  Later  growth  stage. 

233.  Prophase  of  first  maturation  mitosis,  some  of  the  chromosomes  split 

longitudinally. 

234.  Later  stage,  chromosomes  condensing  and  element  x  dividing. 
235-237.  First  maturation  mitosis. 

238.  Division  of  element  x  between  the  two  maturation  divisions. 

239.  Second  maturation  mitosis. 

240.  Anaphase  of  same,  showing  the  element  x  more  deeply  stained  than 

the  chromosomes. 
247.  Young  spermatids ;  element  x  still  conspicuous. 


STEVENS. 


PLATE   VII. 


214 


215 


APHIS  CENOTHERA— SAGITTA  BIPUNCTATA. 


THE  UNIVERSITY  LIBRARY 

UNIVERSITY  OF  CALIFORNIA,  SANTA  CRUZ 

SCIENCE  LIBRARY 

This  book  is  due  on  the  last  DATE  stamped  below 


DEC  3    1969 
MOV  1 4 


50m-4,'69(J7948s8)2477 


